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Category Antibacterial.

      Erythromycin Stearate is the stearic acid salt of Erythromycin, with an excess of Stearic acid. The sum of the percentages of erythromycin A, erythromycin B, and erythromycin C is not less than 55.0 per cent, calculated on the anhydrous basis.

Description White, crystalline powder.

Solubility Practically insoluble in water; soluble in acetone and in methanol. Solutions may be opalescent.

Contra-indication; Warning; Precaution; Additional information See under Erythromycin, p. 99.

Packaging and storage Erythromycin Stearate shall be kept in tightly closed containers, protected from light.

Labelling The label on the container states (1) number of μg per mg; (2) storage condition.

Identification

      A. The infrared absorption spectrum of the contents is concordant with the spectrum obtained from Erythromycin Stearate RS (Appendix 2.1) or with the reference spectrum of Erythromycin Stearate.

      B. Complies with the tests for Identification B and C described under Erythromycin Ethylsuccinate, p. 103

      C. Heat gently 100 mg with 5 ml of 2 M hydrochloric acid and 10 ml of water until the solution boils: oily globules rise to the surface. Cool, remove the fatty layer, heat it with 3 ml of 0.1 M sodium hydroxide, and allow to cool: the solution sets to a gel. Add 10 ml of hot water and shake: the solution froths. To 1 ml of the solution add a 10 per cent w/v solution of calcium chloride: a granular precipitate, insoluble in hydrochloric acid, is produced.

Crystallinity It is crystalline (Appendix 4.14).

Water Not more than 4.0 per cent w/w (Karl Fischer Method, Appendix 4.12). Use 20 ml of methanol containing 10 per cent of imidazole in place of methanol  in the titration vessel. Use 300 mg.

Sulfated ash Not more than 1.0 per cent after ignition at 600º, the charred residue being moistened with 2 ml of nitric acid and 5 drops of sulfuric acid (Method II, Appendix 5.3).

Related substances Carry out the test as described under Assay, using Assay preparation and Standard preparation 2.

      Calculation Calculate the percentage of each related substance having the greatest response, other than erythromycin A, erythromycin B, erythromycin C, erythromycin A enol ether, and pseudoerythromycin A enol ether (retention time relative to erythromycin A peak is about 1.5), in the portion of Erythromycin Stearate taken by the expression:

30(C_S2 P/W)(r_i /r_S2),

in which C_S2 is the concentration, in mg per ml, of Erythromycin RS in Standard preparation 2; P is the designated percentage of erythromycin A in Erythromycin RS; W is the quantity, in mg, of Erythromycin Stearate taken to prepare the Assay preparation; r_i is the peak response of each related compound, other than erythromycin A, erythromycin B, erythromycin C, erythromycin A enol ether, and pseudoerythromycin A enol ether, observed in the chromatogram obtained from Assay preparation; and r_S2 is the erythromycin A peak response in the chromatogram obtained from Standard preparation 2: not more than 3.0 per cent of any individual related compound is found.

      Calculate the percentage of erythromycin A enol ether in the portion of Erythromycin Stearate taken by the expression:

(30/11)(C_S2P/W)(r_E/r_S2),

in which 11 is the response factor for erythromycin A enol ether in relation to that of erythromycin A, r_E is the peak response of the erythromycin A enol ether observed in the chromatogram obtained from Assay preparation, and the other terms are as defined above: not more than 3.0 per cent of erythromycin A enol ether is found.

      Calculate the percentage of pseudoerythromycin A enol ether in the portion of Erythromycin Stearate taken by the expression:

(30/6.6)(C_S2P/W)(r_P/r_S2),

in which 6.6 is the response factor for pseudoerythromycin A enol ether in relation to that of erythromycin A, r_P is the peak response of the pseudoerythromycin A enol ether (retention time relative to the erythromycin A peak is about 1.5) observed in the chromatogram obtained from Assay preparation, and the other terms are as defined above: not more than 3.0 per cent of pseudoerythromycin A enol ether is found.

Assay Carry out the determination as described in the “High-pressure Liquid Chromatography” (Appendix 3.5).

      pH 8.0 buffer Prepare a 2.0 per cent w/v solution of dipotassium hydrogenphosphate, and adjust with phosphoric acid to a pH of 8.0.

      pH 9.0 buffer Prepare a 3.5 per cent w/v solution of dipotassium hydrogenphosphate, and adjust with potassium hydroxide TS or a 10 per cent w/v solution of phosphoric acid, as appropriate, to a pH of 9.0.

      pH 3.5 buffer Adjust 20 ml of pH 8.0 buffer with phosphoric acid to a pH of 3.5.

      Mobile phase Prepare a mixture of 50 volumes of pH 9.0 buffer, 400 volumes of water, 175 volumes of 2- methyl-2-propanol and 30 volumes of acetonitrile, dilute with water to 1000 ml, and mix. Make adjustments if necessary. (Note Use the following solutions promptly, or within 1 day if stored in a refrigerator.)

      Standard preparation 1 Transfer about 40 mg of Erythromycin RS, accurately weighed, to a conical flask, add 5.0 ml of methanol, and swirl to dissolve. Add 5.0 ml of pH 8.0 buffer, and mix.

      Standard preparation 2 Transfer about 6 mg each of Erythromycin RS, Erythromycin B RS, Erythromycin C RS and N-Demethylerythromycin A, all accurately weighed, to a 50-ml conical flask, add 15.0 ml of methanol, and swirl to dissolve. Add 15.0 ml of pH 8.0 buffer, and mix.

      Erythromycin A enol ether solution Dissolve about 5 mg of Erythromycin RS in 1 ml of methanol. Add 5 ml of pH 3.5 buffer, mix, and allow to stand for about 30 minutes

      Assay preparation Transfer about 165 mg of Erythromycin stearate, accurately weighed, to a 100-ml conical flask, add 15.0 ml of methanol, and swirl to dissolve. Add 15.0 ml of pH 8.0 buffer, and mix. Allow the resulting suspension to settle, and pass a portion of the supernatant through a filter having a 0.2-μm or finer porosity. Use the clear filtrate.

      Chromatographic system The chromatographic procedure may be carried out using (a) a stainless steel column (25 cm × 4.6 mm) packed with a rigid, spherical styrene-divinylbenzene copolymer (1000 Å), maintained at a constant temperature of about 70º, (b) Mobile phase at a flow rate of about 2 ml per minute, and (c) an ultraviolet photometer set at 215 nm.

      To determine the suitability of the chromatographic system, chromatograph Standard preparation 2, and record the peak responses as directed under Procedure: the order of elution of the components is N-demethylerythromycin A, erythromycin C, erythromycin A, and erythromycin B; and the resolution factor between N-demethylerythromycin A and erythromycin C peaks is not less than 0.8 and between N demethylerythromycin A and erythromycin A not less than 5.5. Chromatograph Erythromycin A enol ether solution, and adjust the duration of chromatography to include the erythromycin A enol ether peak, which has a retention time of about 4.3 to 4.7 times that of erythromycin A. Chromatograph Standard preparation 1, and record the peak responses as directed under Procedure: the relative standard deviation for replicate injections is not more than 2.0 per cent.

      Procedure Separately inject equal volumes (about 100 μl) of Standard preparation 1, Standard preparation 2 and Assay preparation into the chromatograph, record the chromatograms for a period of time that is adequate to include the erythromycin A enol ether peak, if present, and measure the responses for the major peaks. 

      Calculation Calculate the content of erythromycin A in the portion of Erythromycin Stearate taken by the expression:

30(C_S1P/W)(r_U/r_S1),

in which C_S1 is the concentration, in mg per ml, of Erythromycin RS in Standard preparation 1, P is the designated percentage of erythromycin A in Erythromycin RS, W is the quantity, in mg, of Erythromycin Stearate taken to prepare Assay preparation, and r_U and r_S1, are the erythromycin A peak responses in the chromatograms obtained from Assay preparation and Standard preparation 1, respectively.

      Calculate the content of erythromycin B and erythromycin C in the portion of Erythromycin Stearate taken by the expression:

30(C_S2P/W)(r_U/r_S2),

in which CS2 is the concentration, in mg per ml, of relevant Reference Substance in Standard preparation 2, P is the designated percentage of erythromycin B in Erythromycin C in the relevant Reference Standard, W is the quantity, in mg, of Erythromycin Stearate taken to prepare Assay preparation, and r_U and r_S2 are the peak responses of the relevant analyte in the chromatograms obtained from Assay preparation and Standard preparation 2, respectively. The percentage of erythromycin B is not more than 12.0 per cent; and the percentage of erythromycin C is not more than 5.0 per cent.